Serum klotho associated with thyroid hormone in adults: A population-based cross-sectional research

Background and study aim The klotho protein, a multifunctional protein, has been shown to be associated with a wide range of endocrine diseases and has been linked to thyroid tumourigenesis. However, the relationship between serum klotho levels and thyroid hormones remains poorly understood. This study aimed to explore the correlation between serum klotho levels and thyroid hormones. Methods Data was obtained from the NHANES cycles 2007–2008, 2009–2010, and 2011–2012. A total of 4674 participants were recruited for this study. Statistical analysis was using multiple linear regression analyses, and restricted cubic spline plots (RCS) to investigate the association between serum klotho levels and serum levels of thyroid hormones. Results In the unadjusted covariate model, ln(klotho) significantly positively correlated with tT3, tT4, fT3, tT4/fT4, and tT3/fT3 (all P<0.01) and negatively correlated with TSH, tT4/tT3, and fT4/fT3 (all P<0.05). Furthermore, tT3, tT4, fT3and tT3/fT3 (P < 0.05) were still significant in the adjusted model. And it is worth noting that there is an approximately L-shaped nonlinear relationship between ln(klotho) and fT3,tT3 with a cut-off point of 6.697 (P-non-linear < 0.05). The stratification analysis showed gender and iodine level differences in the relationship between serum Klotho levels and thyroid hormones. Conclusion There is an L-shaped nonlinear relationship between ln(klotho) and fT3, tT3, suggesting that klotho could be involved in the physiological regulation of thyroid function.


Introduction
The thyroid gland is one of the largest endocrine glands in the body and produces thyroid hormones, namely thyroxine (T4) and triiodothyronine (T3) [1].Thyroid hormones act on almost all nucleated cells and play vital roles in the body [2,3].The regulation of thyroid hormones is associated with a variety of substances such as sodium/iodide isotransporter protein (NIS), thyroglobulin (TG), thyroid peroxidase (TPO), monoiodotyrosine (MIT), diiodotyrosine (DIT) and μ-Crystallin (CRYM) [4][5][6][7][8].New studies have found that klotho protein is associated with the development of thyroid tumours [9].However, the association between klotho protein and thyroid hormones has not been investigated.
The klotho protein was first identified by Kuro-o et al. in 1997 [10].The klotho gene encodes a 130 kDa type I single-pass transmembrane glycoprotein called α-Klotho, which has three isoforms (α, β, and γ) [11,12].Klotho proteins have been shown to exert anti-oxidative, anti-inflammatory, and anti-apoptosis effects and regulate calcium and phosphorus metabolism [13].Pawlikowski et al. reported that a low expression of a-Klotho was involved in thyroid tumor formation [14].Previous study has demonstrated that fT3 levels may be positively correlated with sKlotho concentrations [15].In 3T3-L1 adipocytes, T3 significantly increased the expression levels of membrane form of the klotho gene [16].Based on the above studies, it is suggested that klotho protein is associated with thyroid cancer and fT3, however, the correlation between klotho protein and thyroid hormones and exactly how it is associated has not been clarified.
This study used serum klotho and thyroid function data for participants who participated in the National Health and Nutrition Examination Survey (NHANES) cycles 2007-2008, 2009-2010, and 2011-2012.This study explored the association between serum klotho and thyroid function using data from NHANES.

Study population
The National Health and Nutrition Examination Survey (NHANES) is an ongoing nationwide survey conducted every two years to evaluate the health and nutritional status of the non-institutionalized U.S. population.NHANES uses sophisticated multi-stage probability sampling to select participants representing the non-institutionalized civilians in the U.S. Detailed information on survey design and methods was as previously described [17].The NHANES study protocols were approved by the National Center for Health Statistics (NCHS) Research Ethics Review Committee.To ensure the protection of the participants' rights, NHANES has obtained informed written consent from all the individuals involved in the study.A total of 8360 subjects were selected from the NHANES cycles 2007-2008, 2009-2010, and 2011-2012.However, 3637 subjects were excluded due to missing thyroid function test results, and 49 subjects were excluded due to pregnancy.Therefore, 4674 eligible subjects were included in the analysis.A flow chart showing the screening of the study participants is shown in Fig 1.

Measurement of serum thyroid hormones
Blood samples were collected into 15 ml vacutainers, then centrifuged to obtain serum for further biochemical analysis.The total serum T4 (TT4), T3 (TT3), and free T3 (FT3) levels were determined using the competitive binding assay method.Free T4 (FT4) serum levels were determined using a two-step enzyme immunoassay method.The serum thyroid stimulating hormone (TSH) was measured using the third-and second-generation immune enzymatic (sandwich) assay.

Determination of serum klotho levels
Soluble-Klotho acts as an endocrine or paracrine factor for various target organs.The Northwest Lipid Metabolism and Diabetes Research Laboratory, affiliated with the University of Washington, measured the serum klotho levels in whole blood samples using ELISA kits.Prior to the analysis, all samples were frozen at 80˚C.Two parallel holes were made in the ELISA plate to measure the klotho levels of the quality control samples, and the average value was considered as the final concentration.For healthy people, the reference range for serum Klotho levels are between 285.8 to 1638.6 pg/mL with a mean of 698.0 pg/mL.

Statistical analysis
Due to skewness, serum levels of klotho were log-transformed to approximate a normal distribution.The ln(klotho) levels and baseline indicators were categorized by quartiles.Continuous variables were expressed as standard deviation ± mean or median (IQR), and categorical variables were shown as counts (%).Comparisons between groups were made using Pearson's χ2 test for categorical variables and ANOVA and Kruskal-Wallis H-test for continuous variables.Use violin charts to visualize thyroid hormone levels more intuitively under different Klotho groups.Exploring the relationship between klotho and thyroid hormones using multivariate linear regression and multivariate logistic regression (positive outcomes with thyroid hormone levels higher than three-quarters of a digit).Regression analysis was performed using two types of model calibration.Model 1 was not adjusted for any covariates, while Model 2 was adjusted for all baseline covariates.The relationship between ln(klotho) and serum thyroid hormone levels was tested for non-linearity.Restrictive cubic spline graph (RCS) is used to explore the nonlinear relationship between the two, and to find the cutoff point.Subgroup analyses were performed to examine whether the association between serum klotho and thyroid hormones was altered by gender and urinary iodine status.All statistical analyses were performed using the R software (version 4.3.0,R statistical computing base) and IBM SPSS statistical software (version 23.0).Two-tailed P-values of less than 0.05 were regarded as statistically significant.

Anthropometric and biochemical characteristics of the study participants
The baseline information of the study participants after stratification by ln(klotho) quartiles is shown in Table 1.A total of 4674 participants were included in the study after excluding participants who did not meet the eligibility criteria.The results showed that high tT3, tT4, fT3, tT4/fT4, and tT3/fT3 levels were associated with high serum levels of Klotho protein in females, non-Hispanic blacks, and patients with a positive history of smoking and alcohol use, the violin diagram further visualizes and demonstrates this point (Fig 2).However, high serum klotho levels were associated with lower values in height, weight, albumin, serum creatinine, glutathione, triglycerides, and uric acid levels.There was no significant difference between the groups in terms of diabetic patients, hypertensive patients, ghrelin, urinary iodine, urinary creatinine.
The levels of tT3, tT4, fT3, TSH, tT4/fT4, tT3/fT3, tT4/tT3, fT4/fT3 higher than their threequarter quartile were used as a positive outcome and multivariate logistic regression was carried out, and the results are shown in Table 3.In the uncorrected model, klotho protein was a risk factor for elevated tT3, fT3, tT4/fT4, tT3/fT3 (P < 0.05), and after adequate correction of the model, klotho protein was still found to be a risk factor for elevated tT3, fT3, whereas its effect on tT4/fT4, tT3/fT3 was no longer significant.Furthermore, to visualize the relationship between klotho proteins and thyroid hormones, a non-linearity test was first performed, and for variables with non-linear relationships, restriction cubic spline plots (RCS) were fitted, see Fig 3 .Notably, we can find a non-linear relationship between klotho proteins and fT3 and tT3 (P-non-linear < 0.05).And the relationship between the two approximates an L-shape, with the cut-off point of 6.697 in both cases.When the cut-off point is exceeded, the relationship between klotho protein and fT3,tT3 gradually tends to be stable.However, the non-linear test between klotho protein and tT4 and tT3/fT3 was not significant (P-non-linear > 0.05), and the linear regression was sufficiently robust.

Discussion
In this large US population-based study, we found that tT3, tT4, fT3, and tT3/fT3 increased with klotho protein in US adults.ln(klotho) was significantly positively correlated with tT3, fT3, and significantly negatively correlated with TSH, tT4, fT4.After adjusting for covariates, ln(klotho) was still significantly correlated with tT3, fT3, tT4, and tT3/fT3 (P < 0.05).Through https://doi.org/10.1371/journal.pone.0301484.g003multivariate logistic regression, we found that klotho protein may be a risk factor for elevated fT3, tT3.And it is noteworthy that there is an approximately L-shaped nonlinear relationship between ln(klotho) and fT3, tT3 with a cut-off point of 6.697.Furthermore, the relationship was significant at values below the cut-off point and not significant at values above the cut-off points, suggesting the existence of threshold effects in the relationship between ln(klotho) and fT3, tT3.
The synthesis and breakdown of thyroid hormones are associated with the involvement of a variety of substances.Studies have proven that either an excess or deficiency of thyroid hormones can cause serious illness.For example, hypothyroidism is associated with coronary heart disease (CHD), dyslipidemia, atherosclerosis, renal insufficiency, and deficits in fine motor skills [18][19][20][21][22]. On the other hand, hyperthyroidism is linked to autoimmune diseases, heart failure, and atrial fibrillation [23,24].Furthermore, several factors affect thyroid hormone levels, including genetics, exposure to pollutants, autoimmunity, inflammation, and oxidative stress [25].Recent study have identified a possible association between klotho proteins and thyroid hormones [26], but the exact relationship has not been proven.
Klotho, an anti-aging gene, also has anti-inflammatory and anti-oxidative stress effects.In humans, the serum levels of klotho proteins decrease with age from the age of 40.Klotho proteins are highly expressed in the kidneys, cerebral choroid plexus, parathyroid glands, pituitary gland, thyroid gland, aorta, ovaries, and testes [27][28][29].Klotho protein is closely associated with various diseases.Elderly patients with hypertension show significantly lower serum klotho levels than those without hypertension, suggesting that the development of hypertension in the elderly population could be associated with reduced levels of klotho protein [30][31][32].Furthermore, patients with chronic renal failure show significantly reduced levels of klotho proteins in the renal tissue, which could be explained by the fact that decreased expression of exacerbation exacerbates interstitial fibrosis [33].In addition, decreased expression of the klotho protein is involved in the development of chronic obstructive pulmonary disease and Alzheimer's disease [34].Furthermore, klotho overexpression was shown to significantly reduce follicular thyroid cancer FTC133 and FTC238 cells proliferation and enhance apoptosis [35].Qiong Wu et al. demonstrated that klotho inhibited cell proliferation in a RET fusion model of PTC by inhibiting the Wnt/β-linked protein pathway [36].The anti-inflammatory and anti-oxidative stress effects of klotho protein could affect thyroid hormones.However, no studies have investigated the correlation between serum klotho levels and thyroid hormones.
In our study, we found that klotho protein may be a risk factor for elevated tT3, fT3, and the results showed that there was a threshold effect in the relationship between ln (klotho) and fT3, tT3.More notably, the cut-off points were both 6.697.By calculation, we found that the mean fT3 value corresponding to the cut-off point was 116.5 ng/dL and the mean tT3 value was 3.1325 nf/dL, which were both within the normal range.We speculate that when ln(klotho) < 6.697, the trend of fT3 and tT3 increasing with it is significant, while when ln(klotho) is greater than 6.697, the trend of fT3 and tT3 increasing with it is no longer obvious, and the specific reasons need to be verified by further mechanism exploration.
This study showed gender disparities in the relationship between serum klotho levels and thyroid hormones, with males having a lower association between ln(klotho) and fT3 than females.Furthermore, the associations between ln(klotho) and tT3, tT4, tT4/fT4, and tT3/fT3 were significant in males but not in females.Previous studies have shown that estrogen increases the expression of thyroglobulin, thyroid peroxidase activity, and iodine uptake, and regulates TSH levels [37][38][39][40].In addition, estrogen modifies the activity and expression of dual oxidase 2 (DUOX2) and nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4), thus influencing the thyroid redox state [41].Therefore, klotho protein may have a considerably lower effect on thyroid hormones than estrogen, indicating that male adults may show more effects of Klotho protein on thyroid hormones than females.However, further studies are needed to show the potential pathways behind the gender disparities between serum klotho protein levels and thyroid hormones.
In addition, previous studies have shown that iodine deficiency and iodine overload could cause thyroid dysfunction since iodine is a micronutrient necessary for the synthesis of thyroid hormones.The present study showed a significantly positive correlation between ln(klotho) and tT3, fT3, tT4/fT4, and tT3/fT3 in participants with urinary iodine >100 ug/L.In contrast, ln(klotho) was significantly positively correlated with tT3 in participants with urinary iodine <100 ug/L.This phenomenon could be explained by the fact that iodine deficiency causes a decrease in circulating TT3 and TT4, resulting in increased TSH release from the pituitary gland through a classical negative feedback mechanism [42], which could diminish the effects of klotho protein on thyroid hormones.
This study had some limitations.First, as this paper is a cross-sectional study, causality cannot be inferred and future longitudinal studies are needed to determine the predictive value of Klotho in thyroid disorders.Secondly, most of the data were obtained from singlesite measurements, and without data on concentration changes, our results can only reflect the relationship between klotho and thyroid hormones at the initial stage of the study.Finally, despite our attempts to adjust for potential confounders as much as possible, there remain a number of unknown or unmeasured confounders that may also play a role in the pathogenesis of thyroid disease.Therefore, multiple measurements in a long-term follow-up study would likely show the association between serum klotho protein levels and thyroid hormones.
In conclusion, the present study demonstrated a correlation between klotho protein levels and thyroid function in U.S. adults.Furthermore, the relationship between klotho protein levels and thyroid hormones is more pronounced in men and adults with adequate iodine levels.However, large prospective studies are required to generalize the findings of this study.

Fig 3 .
Fig 3. Restricted cubic spline plots between ln(Klotho) and tT3, fT3, tT4, tT3/fT3, with the number of knot nodes calculated as 4 according to the minimum AIC criterion,and the vertical line indicates the cutoff point.The model is full-adjusted (corrected for all covariates in the baseline).